ABSTRACT Methionine adenosyltransferase (MAT) is an essential cellular enzyme that catalyzes the formation of S- adenosylmethionine (SAMe), the principal biological methyl donor and in liver, precursor of the key antioxidant glutathione (GSH). In mammals, two different genes, MAT1A and MAT2A, encode for two homologous MAT catalytic subunits, ?1 and ?2, respectively. MAT1A is primarily expressed in normal liver. Majority of patients with chronic liver disease have decreased expression and activity of MAT1A-encoded isoenzymes. We found MAT1A expression and hepatic SAMe levels are reduced in alcoholic hepatitis patients. While MAT isoenzymes are widely acknowledged for catalyzing cytosolic SAMe biosynthesis, our recent works have uncovered highly novel aspects of their functions. In addition to cytosol and nucleus, our preliminary data indicate MAT?1 is also present in the mitochondrial matrix to regulate mitochondrial function. This is important as we found hepatocytes lack the mitochondrial SAMe transporter SLC25A26. Using immunoprecipitation (IP) followed by mass spectrometry, we have identified many mitochondrial proteins and cytochrome P450 2E1 (CYP2E1) as MAT?1-interacting proteins. Our preliminary data show that MAT?1 negatively regulates CYP2E1 expression mainly at the protein level via methylation, which has not been reported. Importantly, MAT?1 mitochondrial targeting is impaired in murine and human alcoholic liver disease (ALD) and we propose two novel mechanisms that may cooperate in causing this impairment. The current proposal tests the central hypothesis that MAT?1 provides the SAMe source within the hepatocyte's mitochondrial matrix and impairment in MAT?1 mitochondrial targeting in ALD plays a key role in the pathogenesis of ALD. The corollary hypothesis is that MAT?1 maintains hepatocyte mitochondrial function in part by suppressing CYP2E1 expression. Here we follow up these novel findings in three specific aims: 1) examine how MAT?1 targets the mitochondria and why its targeting is impaired in ALD. We will elucidate how MAT?1 targets the mitochondria and mechanisms of its impaired targeting in ALD. We will test the novel hypothesis that this is due to 1) increased MAT?1 sumoylation and 2) increased interaction of MAT?1 with PIN1 (a peptidyl-prolyl cis- trans isomerase that recognizes a specific phosphorylated motif), 2) examine how MAT?1 regulates CYP2E1 protein expression. We will examine how MAT?1 regulates CYP2E1 protein stability at the molecular level and identify CYP2E1 residues and interacting proteins that participate in this process, 3) examine the role of mitochondrial MAT?1 and MAT?1-regulated CYP2E1 in ALD. We will test the novel hypothesis that preventing MAT?1 sumoylation or interaction with PIN1 will protect against reduced mitochondrial MAT?1 content and injury in ALD. We will also examine whether MAT?1 will protect against ALD by methylating CYP2E1 at R379 to enhance its degradation. If successfully accomplished, these studies will change existing paradigms on MAT?1 biology, CYP2E1 regulation and may uncover druggable targets for ALD.